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Mexedrone is a synthetic cathinone structurally related to mephedrone, which belongs to the class of N-alkyl cathinone derivatives, whose metabolic profile has not been fully clarified yet. This study considers the in vitro phase I metabolism of mexedrone, to pre-select the most appropriate marker(s) of intake. Mexedrone was incubated in the presence of either human liver microsomes or single recombinant CYP450 isoforms. The metabolic profile was outlined by ultra-high-performance liquid chromatography coupled to both high- and low-resolution mass spectrometry. In detail, the phase I metabolic profile of mexedrone was initially defined by a time-of-flight analyzer, while the chemical structures of the detected metabolites and the potential presence of minor metabolites were subsequently studied by tandem mass spectrometry, using a triple quadrupole analyzer. The main phase I metabolic reactions were hydroxylation and N- and O-dealkylation. The CYP450 isoforms most involved were CYP2C19, responsible for the formation of both hydroxylated and dealkylated metabolites, followed by CYP2D6 and CYP1A2, involved in the hydroxylation reactions only. Finally, a significant fraction of mexedrone unchanged was also detected. Based on this evidence, the most appropriate markers of intake are mexedrone unchanged and the hydroxylated metabolites.

This article reports the results obtained from the investigation of the influence of miconazole administration on the physiological fluctuation of the markers of the steroid profile included in the “steroidal module” of the Athlete Biological Passport. Urines collected from male Caucasian subjects before, during, and after either systemic (i.e., oral and buccal) or topical (i.e., dermal) treatment with miconazole were analyzed according to validated procedures based on gas chromatography coupled to tandem mass spectrometry (GC–MS/MS) (to determine the markers of the steroid profile) or liquid chromatography coupled to MS/MS (LC–MS/MS) (to determine miconazole urinary levels). The results indicate that only after systemic administration, the markers of the steroid profile were significantly altered. After oral and buccal administration, we have registered (i) a significant increase of the 5α‐androstane‐3α,17β‐diol/5β‐androstane‐3α,17β‐diol ratio and (ii) a significant decrease of the concentration of androsterone, etiocholanolone, 5β‐androstane‐3α,17β‐diol, and 5α‐androstane‐3α,17β‐diol and of the androsterone/etiocholanolone, androsterone/testosterone, and 5α‐androstane‐3α,17β‐diol/epitestosterone ratios. Limited effects were instead measured after dermal intake. Indeed, the levels of miconazole after systemic administration were in the range of 0.1–12.5 μg/ml, whereas after dermal administration were below the limit of quantification (50 ng/ml). Significant alteration started to be registered at concentrations of miconazole higher than 0.5 μg/ml. These findings were primarily explained by the ability of miconazole in altering the kinetic/efficacy of deglucuronidation of the endogenous steroids by the enzyme β‐glucuronidase during the sample preparation process. The increase of both incubation time and amount of β‐glucuronidase was demonstrated to be effective countermeasures in the presence of miconazole to reduce the risk of uncorrected interpretation of the results., The results obtained from the investigation of the influence of miconazole administration on the parameters included in the “steroidal module” of the Athlete Biological Passport were presented. After oral and buccal administration of miconazole, the levels of the markers of the steroid profile were significantly altered, whereas no effects were registered after topical administration. These findings were primarily explained by the effect of miconazole on the kinetics/efficacy of the enzymatic hydrolysis during the sample preparation process.

The detection of clostebol misuse in sports has been growing recently, especially in Italy, due to the ample availability of pharmaceutical formulations containing clostebol acetate (Trofodermin®) and the use of more sensitive instrumentation by the antidoping laboratories. Most of these cases have been claimed to be related to a nonconscious use of the drug or through contact with relatives or teammates using it. We have investigated, through the application of the well-known and currently used gas chromatographic mass spectrometric procedures, the likelihood of these allegations and have demonstrated that after a single transdermal administration of 5 mg of clostebol acetate and a transient contact with the application area, it is possible to generate adverse analytical findings in antidoping controls. We have reviewed the Phase I and Phase II clostebol metabolism in order to generate evidences that may help the sport authorities reviewing these cases. The main clostebol metabolite (4-chloro-androst-4-en-3α-ol-17-one, M1) generally used at the screening level as well as other three metabolites (M2-M4) are mainly excreted as glucuronides, whereas M5 (4ζ-chloro-5ζ-androstan-3β-ol-17-one) is predominantly excreted as sulfate. Neither the 5α-reductases activity (impaired by the presence of the chlorine in C4) nor specific sulfotransferases present in the skin allowed a clear distinction of the administration route. Studies with a larger number of volunteers and probably investigating another physiological fluid allowed in antidoping such as blood are needed for a deeper investigation. It is not unreasonable to establish a reporting level for M1, maybe creating some false negatives but excluding nonintentional doping scenarios.

Twenty-two pharmaceutical formulations containing prednisolone or prednisone commercially available in Italy, Belgium, Spain, Brazil, and India were analyzed through a specific gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) method. All of them showed typical non-endogenous δ13 C values, except for the Belgian nasal spray, Sofrasolone®, with a less depleted 13 C content (-17.84 ± 0.18‰). Observational studies were performed on two volunteers in therapy with Sofrasolone® to confirm the applicability of the method and to suggest adequate interpretation criteria also in the case of drugs with less negative δ13 C values. Urine samples were collected before, during, and within the 36 hours after the administration of the spray. Both δ13 C values and urinary concentrations of prednisolone and prednisone were evaluated. All samples were subjected to an adequate pre-treatment (enzymatic hydrolysis, liquid/liquid extraction, and two sequential HPLC steps) before injection to the GC-C-IRMS instrument, according to the method recently developed and validated in our laboratory. Pregnanediol (PD), tetrahydro-11-deoxycortisol (THS), and pregnanetriol (PT) were selected as endogenous reference compounds (ERC). The excretion profile was estimated through liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method used routinely for the quali-quantitative detection of glucocorticoids. δ13 C values and urinary levels of prednisolone and prednisone were also determined after the intake of one single vial of Sintredius®, a prednisolone oral formulation with a conventional more negative δ13 C value (-29.28 ± 0.25‰). Finally, the potential masking effect that combined therapy with Sofrasolone® and Sintredius® could induce on the IRMS findings was investigated.

Anabolic androgenic steroids (AAS) are prohibited as performance-enhancing drugs in sports. Among them, testosterone and its precursors are often referred to as “pseudoendogenous” AAS, that is, endogenous steroids that are prohibited when administered exogenously. To detect their misuse, among other methods, the World Anti-Doping Agency-accredited laboratories monitor the steroid profile (concentrations and concentration ratios of endogenous steroids, precursors and metabolites) in urine samples collected from athletes in and out of competition. Alterations in steroid profile markers are used as indicators for misuse of anabolic steroids in sports. Therefore, especially their metabolic pathways with possible interactions are crucial to elucidate. As steroid metabolism is very complex, and many enzymes are involved, certain non-prohibited drugs may influence steroid metabolite excretion. One important group of steroid-metabolizing enzymes is aldo–keto reductases (AKRs). An inhibition of them by non-steroidal anti-inflammatory drugs (NSAIDs), which are neither prohibited nor monitored, but frequently used drugs in sports, was demonstrated in vitro. Thus, this work aims to investigate the influence of NSAID intake on the urinary steroid profile. Kinetic and inhibitory studies were performed using 5α-dihydrotestosterone as substrate. The results obtained from in vitro experiments show that ibuprofen inhibits AKR1C2 and thus influences steroid biotransformation. For in vivo investigations, urine samples prior, during and postadministration of ibuprofen were analyzed using routine methods to monitor the steroid profile. Changes in markers of the steroid profile of volunteers were observed. The combination of in vitro and in vivo results suggests that monitoring of ibuprofen may be useful in doping control analysis. The presented work illustrates the importance to consider co-administration of (non-prohibited) drugs during antidoping analysis. Intake of multiple substances is likely leading to interfering effects. Divergent results in antidoping analysis may therefore be observed and misinterpretation of analytical data may occur. Similar considerations may be appropriate for other fields of forensic applications.

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail.Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite.The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17β-methyl-18-nor-androsta-4,13-dien-3β-ol. It may serve as additional confirmatory metabolite.It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general.

5α-reductase inhibitors (5-ARIs) are considered by the World Anti-doping Agency as potential confounding factors in evaluating the athlete steroid profile, since they may interfere with the urinary excretion of several diagnostic compounds. We herein investigated 5α-reductase inhibitors from a different perspective, by verifying their influence on the carbon isotopic composition of 5α- and 5β-reduced testosterone and nandrolone metabolites. The GC-C-IRMS analysis was performed on a set of urine samples collected from three male Caucasian volunteers after the acute and chronic administration of finasteride in combination with the intake of 19-norandrostenedione, a nandrolone precursor. The excretion and the isotopic profile of androsterone (A), etiocholanolone (Etio) 5α-androstane-3α,17β-diol (5αAdiol), and 5β-androstane-3α,17β-diol (5βAdiol) were determined as well as those of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). Pregnanediol (PD) and pregnanetriol (PT) were also measured as endogenous reference compounds to define the individual endogenous isotopic profile. Our results confirmed the impact of finasteride, especially if chronically administered, on the enzymatic pathway of testosterone and nandrolone, and pointed out the influence of 5-ARIs on δ13 C values of the selected target compounds determined in the IRMS confirmation analysis.

Background: Exercise-associated immune response plays a crucial role in the aging process. The aim of this study is to investigate the effect of sport intensity on cytokine levels, oxidative stress markers and telomere length in aging elite athletes. Methods: In this study, 80 blood samples from consenting elite athletes were collected for anti-doping analysis at an anti-doping laboratory in Italy (FMSI). Participants were divided into three groups according to their sport intensity: low-intensity skills and power sports (LI, n = 18), moderate-intensity mixed soccer players (MI, n = 31), and high-intensity endurance sports (HI, n = 31). Participants were also divided into two age groups: less than 25 (n = 45) and above 25 years old (n = 35). Serum levels of 10 pro and anti-inflammatory cytokines and two antioxidant enzymes were compared in age and sport intensity groups and telomere lengths were measured in their respective blood samples. Results: Tumor necrosis factor-alpha (TNF-α) was the only cytokine showing significantly higher concentration in older athletes, regardless of sport intensity. Interleukin (IL)-10 increased significantly in HI regardless of age group, whereas IL-6 concentration was higher in the older HI athletes. IL-8 showed a significant interaction with sport intensity in different age groups. Overall, significant positive correlations among levels of IL-6, IL-10, IL-8 and TNF-α were identified. The antioxidant catalase activity was positively correlated with levels of TNF-α. Telomere length increased significantly with sport intensity, especially in the younger group. Conclusion: HI had longer telomeres and higher levels of pro- and anti-inflammatory cytokines, suggesting less aging in HI compared to low and moderate counterparts in association with heightened immune response. Investigation of the functional significance of these associations on the health and performance of elite athletes is warranted.

Rationale Isoflavones are a group of flavonoids that may be of interest in sport doping because they can be used by athletes in the recovery periods after the administration of anabolic steroids, with the aim of increasing the natural production of luteinizing hormone (LH) and, consequently, the biosynthesis of endogenous androgens. Methods The in vivo metabolism of methoxyisoflavone (5-methyl-7-methoxyisoflavone) and ipriflavone (7-isopropoxyisoflavone), respectively present in a dietary supplement and in a pharmaceutical preparation, was investigated. The study was carried out by the analysis of urinary samples collected from male Caucasian subjects before, during and after the oral administration of methoxyisoflavone or ipriflavone. After enzymatic hydrolysis and liquid-liquid extraction, all urinary samples were analyzed by gas chromatography/quadrupole time-of-flight (qTOF MS system/qTOF) electron ionization mass spectrometry (EI-MS). Results Eight metabolites of methoxyisoflavone and six metabolites of ipriflavone were isolated. The corresponding accurate mass spectra are specific for isoflavone structures and revealed also a retro-Diels-Alder fragmentation. Conclusions When excreted in large amounts, the urinary metabolites of methoxyisoflavone and ipriflavone can be traced to potential confounding factors in doping analysis. As methoxyisoflavone and ipriflavone have been shown to inhibit the enzyme aromatase, thus interfering with the normal metabolic pathways of testosterone, the detection of their intake, by screening for the presence of their main metabolites in urine, might be helpful in routine doping control analysis.

Adrenal and gonadal disorders are very often coupled, due to common etiology or pathophysiology. We present the development, validation and application of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of androgens (androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), and dehydroepiandrosterone sulfate (DHEA-S)), estrogens (estrone (E1), estradiol (E2), estriol (E3)), glucocorticoids (cortisol (F), cortisone (E), corticosterone (B), 11-deoxycortisol (S), 21-deoxycortisol (21DF), 11-deoxycorticosterone (11DB)), and progestagens (progesterone (P4), 17α-hydroxyprogesterone (17OHP4) and 17α-hydroxypregnenolone (17OHP5)) in human serum for clinical use. Samples (250 &[mu]L of matrix) spiked with isotopic labelled internal standards were extracted with tert-butylmethyl ether (TBME) prior to LC-MS/MS analysis. The chromatographic separation of the underivatized endogenous steroids was achieved on a reversed-phase column (C18 Zorbax Eclipse Plus) using a methanol-water gradient. The LC column was coupled to a triple quadrupole mass spectrometer equipped with an electrospray (ESI) source operating both in positive and in negative mode, with acquisition in multiple reaction mode. The method was validated using surrogated matrices and human serum samples. The proposed method was proven to be specific for all the considered steroids; and linearity was also assessed (R2 > 0.99) in the ranges of quantification investigated. The lower limits of quantification (LLOQs) were in the range of 10 - 400 pg/mL depending on the target steroid. Accuracy was in the range 80 - 120% for all the target compounds, the extraction recovery was higher than 65% for all the steroids considered and no remarkable matrix effect, expressed in terms of ion enhancement and ion suppression, was observed. To test the reliability of the developed and validated method, the analysis of serum samples collected from ten healthy subjects (5M/5F) was performed. In the clinical settings there is a growing need to develop accessible methods for full steroid hormone profiling. The dynamic link between steroidogenic glands and liver enzymatic processing (activation and clearance) attributes to the profile a much greater clinical meaning than a set of individually measured hormones. The presented method can be used to identify trajectories of deviation from the concentration normality ranges applied to disorders of the gonadal and adrenal axes.

In this work we have investigated the influence of the intake of two synthetic isoflavones, methoxyisoflavone and ipriflavone, on the urinary concentration of endogenous steroids, and on their relative ratios, of doping relevance. Specifically, the concentrations of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5α-androstan-3α,17α-diol (5αAdiol), 5β-androstan-3α,17α-diol (5βAdiol), and the ratios T/E, A/T, A/Etio, 5αAdiol/5βAdiol, 5αAdiol/E, were considered, in the framework of the Steroidal Module of the Athlete Biological Passport (ABP). The above set of parameters were complemented by the urinary levels of luteinizing hormone (total LH) and the ratio between T and LH (T/total LH), to assess the possible effects on the biosynthesis of the mentioned steroids. Five healthy Caucasian male volunteers were selected for the study. Urine samples were collected before and during the administration of (i) methoxyisoflavone (Methoxyisoflavone, MyProtein) and (ii) ipriflavone (Osteofix ®, Chiesi Farmaceutici). For the analysis of the urinary steroid profile, after enzymatic hydrolysis with β-glucuronidase from Escherichia Coli (E. Coli) and liquid–liquid extraction with tert-buthylmethyl ether, all samples were analyzed by gas chromatography coupled to tandem mass spectrometry (GC–MS/MS), while for the determination of total LH all urine samples were directly analyzed by a chemiluminescent immunometric assay technique (Siemens Immulite 2000 LH). Our results show that the administration of either methoxyisoflavone or ipriflavone causes an alteration of the urinary concentrations and concentration ratios of the investigated steroids, in the range 55–80% from the baseline values. Furthermore, an oversecretion of LH after the daily intake of methoxyisoflavone or ipriflavone was also recorded in all volunteers, corresponding to an increase in the biosynthesis and excretion of T and some of its metabolites. These changes trigger a disregulation in the pattern of urinary excretion of the steroids included in the Steroidal Module of the ABP, which makes more difficult the interpretation of the longitudinal steroid profile based on the definition of individual normality ranges for each athlete. Our data are also consistent with previous evidence regarding the in vitro effects of natural and synthetic isoflavones, suggesting that their monitoring in doping control routine analysis would be very beneficial for the result management activities.

Transcranial Direct Current Stimulation (tDCS) is a non-invasive brain stimulation that may enhance mental and physical performance in sports, representing a potential new form of doping (“brain doping” or “electromagnetic doping”). This study aims to identify diagnostic biomarkers for detecting the possible abuse of tDCS in sport. Brain-Derived Neurotrophic Factor (BDNF) and other neurotrophins (NT, such as beta nerve growth factor, NGF) were pre-selected as potential candidates since their serum values have been observed to change following tDCS. Neurotrophins were measured using ELISA assays in 92 serum samples collected from elite athletes, classified by sex (males = 74; females = 18), age (range 17–25 n = 27, 26–35 n = 36, and over 35 n = 14; age not known n = 15), type of sports practiced (endurance n = 74; power n = 18), and type of sample collection (“in competition” n = 24; “out of competition” n = 68). Single nucleotide polymorphisms (rs6265, rs11030099, and rs11030100) were genotyped on 88 samples to determine their influence on the analytes' basal levels. Athletes older than 35 presented higher BDNF values than younger individuals (p < 0.05). Samples collected “in competition” showed higher BDNF concentrations than those collected “out of competition” (p < 0.05). The studied polymorphisms appeared to affect only on proBDNF, not altering BDNF serum concentrations. NT-3 and NT-4 were poorly detectable in serum. Our results suggest that BDNF can be considered as a first biomarker to detect the abuse of tDCS in sport doping. Further studies are necessary to assess whether proBDNF and beta NGF can also be considered suitable biomarkers to detect the recourse to electromagnetic brain stimulation in sports, especially in the case their serum levels can be monitored longitudinally. To the best of our knowledge, this is the first study aimed to pre-select serum biomarkers to identify the use of tDCS, and represents the first step toward the development of an indirect strategy, preferably based on the longitudinal monitoring of individual data, for the future detection of “brain doping” in sports.

Metandienone and methyltestosterone are orally available anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. After the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone (“Oral Turinabol”), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol and its 5α-analog, were identi-fied following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5β-metabolite was detected. Additionally, 3α,5β-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new hypotheses on the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring and the participation of different enzymes.

Background Over the counter preparations containing Saw palmetto and Pygeum africana extracts are commonly used in the treatment of benign prostatic hyperplasia with lower urinary tract symptoms. Their effects are likely due to their anti-androgenic activity, involving the interaction with type 1 and type 2 isoenzymes of the 5α-reductase family. Hypothesis/purpose For effects similar to the above, the inhibitors of 5α-reductase (e.g., finasteride) are classified as confounding factors in the evaluation of the urinary steroid profile in sport doping. We aimed to verify whether the oral intake of products containing Saw palmetto and Pygeum africana could affect the urinary concentration of a series of steroids, not limited to those included in the “steroidal module” of the Athlete Biological Passport (ABP). Study design We have followed the effects of a five-day administration of Saw palmetto (320 mg) and Pygeum africana (100 mg) to three non-smokers male subjects on the urinary parameters constituting the “steroidal module” of the ABP, and on the concentration of additional steroid markers. In parallel, we have also measured the urinary concentration of β-sitosterol, which is the active component of Saw palmetto and Pygeum Africana. Methods Sample collection was performed five days before starting the controlled administration study to obtain the baseline urinary steroid pattern for each subject. After this period, the three volunteers took a dose of Saw palmetto (320 mg) and Pygeum africana (100 mg) formulation orally, once a day, for five consecutive days. Urine samples were collected at different times of the day and stored at −20 °C until analysis. Samples were analyzed by a previously developed and validated method based on gas chromatography coupled to tandem mass spectrometry (GC–MS/MS). Results Our results show that the intake of a single oral dose of Saw palmetto and Pygeum africana for five consecutive days leads to a broader data dispersion of the target steroids' concentration levels, and, consequently, to a variation of the corresponding individual normality ranges. Conclusion The intake of dietary supplements containing Saw palmetto and Pygeum africana could be a source of interference in the correct longitudinal evaluation of the individual steroid profile, as described in the framework of ABP. We believe their urinary levels should be monitored as it is presently done for other confounding factors of the steroidal module of the ABP.

The detection of 19-norsteroids abuse in doping controls currently relies on the determination of 19-norandrosterone (19-NA) by gas chromatography-tandem mass spectrometry (GC-MS/MS). An additional confirmatory analysis by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) is performed on samples showing 19-NA concentrations between 2.5 and 15 ng/ml and not originated from pregnant female athletes or female treated with 19-norethisterone. 19-Noretiocholanolone (19-NE) is typically produced to a lesser extent as a secondary metabolite. The aim of this work was to improve the GC-C-IRMS confirmation procedure for the detection of 19-norsteroids misuse. Both 19-NA and 19-NE were analyzed as target compounds (TCs), whereas androsterone (A), pregnanediol (PD), and pregnanetriol (PT) were selected as endogenous reference compounds (ERCs). The method was validated and applied to urine samples collected by three male volunteers after the administration of nandrolone-based formulations. Before the instrumental analysis, urine samples (

© Copyright © 2020 Sohail, Al-Mansoori, Al-Jaber, Georgakopoulos, Donati, Botrè, Sellami and Elrayess. Objectives: Circulating cytokines and oxidative stress markers vary in response to different exercise regimens. This study aims to compare the immune-inflammatory and oxidative stress profiles of elite athletes from different sport disciplines as potential biomarkers of muscle damage, and cardiovascular demand. Methods: Serum samples from 88 consented elite male athletes from different sports disciplines (aquatics, n = 11, athletics, n = 22, cycling, n = 19, football, n = 28 and weightlifting, n = 8) collected at the anti-doping lab in Italy were screened for 38 cytokines and oxidative stress markers. Comparisons were made between different level of power, cardiovascular demand (CD) and endurance, as well as among the sport types. Results: The anti-inflammatory interleukin (IL)-10 was higher (p = 0.04) in moderate power compared with the high power group. Conversely, superoxide dismutase (SOD; p = 0.001) and malondialdehyde (MDA; p = 0.007) levels were greater in the higher power groups compared with the lower power counterpart. Among athletes who belong to different CD ranks, IL-1β and monocyte chemoattractant protein-1(MCP1) levels were higher (p = 0.03) in the low CD-rank group compared with high CD counterpart, whereas, SOD levels were higher (p = 0.001) in high and moderate CD-rank groups compared to low counterpart. For endurance groups, IL-10 and macrophage inflammatory protein (MIP)-1beta were increased (p = 0.03) in low/moderate endurance compared with the high endurance group. Finally, MIP1-beta, SOD and catalase varied significantly among the sports groups. Conclusion: Specific markers of inflammation and oxidative stress are associated with different sports disciplines and could be utilized as potential biomarkers of athletes’ health, performance, and recovery from injury. We would like to thank Qatar National Research Fund (QNRF) for funding this project.

Predictive models have been developed for the early identification of novel anabolic androgenic steroids and to obtain information on their molecular structure. To this purpose, gas-chromatographic and mass spectrometric characteristic parameters of 136 anabolic androgenic steroids have been specifically considered. Starting from Principal Component Analysis, different chemometric methods were applied, such as classification and clustering techniques, outlining a spectral and structural characterization for each steroid subclass, and considering the contribution of more than 30 variables. Mass spectrometric data on the TMS-derivatives of the target steroids were obtained by gas chromatography coupled to quadrupole-time of flight mass spectrometry using electron ionization. Steroids included in the training set were grouped in 5 subclasses according to their structural similarity, and the experimental data, processed by the chemometric models, allowed the identification of class-specific common fragments and spectral trends. The results of this study, validated on a test set of 21 steroids, have confirmed that the proposed approach allows tracing novel “designer anabolic steroids”, including those previously unknown new structures that may have been designed and illicitly synthesized to be invisible to the current anti-doping tests.

The detection of the abuse of pseudo-endogenous steroids in sport is articulated in two different levels: an initial testing procedure, based on the longitudinal evaluation of the urinary androgenic steroid profile by gas-chromatography mass spectrometry (GC-MSn), and a confirmation analysis, based on the differentiation between the endogenous and exogenous origin of the pseudo-endogenous steroids by gas-chromatography coupled to isotopic ratio mass spectrometry (GC/C/IRMS). The abuse of pharmaceutical preparations displaying a carbon isotopic composition values within a range similar to those reported for endogenous urinary steroids makes more difficult the application of GC/C/IRMS technique. To overcome this limitation, the direct detection of an intact synthetic anabolic steroid ester in blood matrices (plasma and/or serum) could supply the unequivocal proof of exogenous administration of pseudo-endogenous steroids. Here we are presenting a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of 14 testosterone (T) esters and 2 nandrolone (Nand) esters in human serum. Sample pre-treatment consisted of protein precipitation, liquid-liquid extraction and derivatization. The formation of three different derivatives (oxime derivatives, Girard P and Girard T hydrazones) is considered, in order to guarantee an improvement in the detection capability of the assay with respect to underivatized compounds. Once the most suitable derivative was selected, the method was validated, according to the World Anti-Doping Agency (WADA) criteria, in terms of specificity, linearity, limit of detection (LOD), extraction recovery, matrix effect (ion suppression/enhancement), carry over and autosampler stability. The formation of Girard P hydrazones of T and Nand esters provides the best results compared to the underivatized compounds, oxime and Girard T derivatives, respectively. The presented analytical method is specific for all considered compounds and linear in the range of concentrations investigated (0.25-10 ng/mL). The LODs are between 0.03 and 0.30 ng/mL, the extraction recovery higher than 70 % for all esters and no remarkable matrix effect, expressed in terms of ion enhancement and ion suppression, was observed. Finally, the developed and validate method was applied in the analysis of serum samples collected after the administration of a single dose (40 mg, 1 capsule) of testosterone undecanoate (Andriol ®) demonstrating its applicability.

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Background The genetic predisposition to elite athletic performance has been a controversial subject due to the underpowered studies and the small effect size of identified genetic variants. The aims of this study were to investigate the association of common single-nucleotide polymorphisms (SNPs) with endurance athlete status in a large cohort of elite European athletes using GWAS approach, followed by replication studies in Russian and Japanese elite athletes and functional validation using metabolomics analysis. Results The association of 476,728 SNPs of Illumina DrugCore Gene chip and endurance athlete status was investigated in 796 European international-level athletes (645 males, 151 females) by comparing allelic frequencies between athletes specialized in sports with high (n = 662) and low/moderate (n = 134) aerobic component. Replication of results was performed by comparing the frequencies of the most significant SNPs between 242 and 168 elite Russian high and low/moderate aerobic athletes, respectively, and between 60 elite Japanese endurance athletes and 406 controls. A meta-analysis has identified rs1052373 (GG homozygotes) in Myosin Binding Protein (MYBPC3; implicated in cardiac hypertrophic myopathy) gene to be associated with endurance athlete status (P = 1.43 × 10-8, odd ratio 2.2). Homozygotes carriers of rs1052373 G allele in Russian athletes had significantly greater VO2 max than carriers of the AA + AG (P = 0.005). Subsequent metabolomics analysis revealed several amino acids and lipids associated with rs1052373 G allele (1.82 × 10-05) including the testosterone precursor androstenediol (3beta,17beta) disulfate. Conclusions This is the first report of genome-wide significant SNP and related metabolites associated with elite athlete status. Further investigations of the functional relevance of the identified SNPs and metabolites in relation to enhanced athletic performance are warranted.

This brief commentary presents an overview of the potential utility of metabolomics-based approaches in doping analysis, with particular emphasis on areas where the current analytical procedures would most benefit from an improvement of their efficacy. Metabolomics, first defined by O Fiehn at the beginning of this millennium as “a comprehensive and quantitative analysis of all metabolites” of a given biological system [1], has not been considered in the field of doping analysis until very recently.With the exception of the monitoring of the urinary steroid profile, which is de facto a form of ‘targeted steroidomics’, metabolomics-based procedures are not yet part of the routine analytical activity of the antidoping laboratories accredited by the World Anti-Doping Agency (WADA). We aim to supply a critical view on the potential utility of metabolomics-based approaches in doping analysis, outlining how they could complement the analytical procedures already in place at the WADA accredited laboratories, and what is still needed to ensure their actual application in doping analysis.

Although the metabolism of methyltestosterone (MT) has been extensively studied since the 1950s using different techniques, the aim of this study was to investigate the hydroxylation in positions C2, C4 and C6 after in vitro experiments and in vivo excretion studies using gas chromatography time-of-flight (GC/TOF) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The results could be influenced by the mass spectrometric analyser used.Incubations were carried out with human liver microsomes and six enzymes belonging to the cytochrome P450 family using MT as a substrate. The trimethylsilyl derivatives of the samples were analysed using GC/TOF and GC/MS/MS once the correct MS/MS transitions had been selected, mainly for 6-hydroxymethyltestosterone (6-OH-MT) to avoid artefact interferences. A urinary excretion study was then performed after the administration of a 10 mg single oral dose of MT to a volunteer.The formation of hydroxylated metabolites of MT in the C6, C4 and C2 positions after both in vitro and in vivo experiments was observed. Sample evaluation using GC/TOF showed an interference for 6-OH-MT that could only be resolved in GC/MS/MS by monitoring specific transitions. The transitory detection of these hydroxylated metabolites in urine agrees with previous investigations that had described this metabolic route as being of little significance.In doping analysis, the formation of 4-hydroxymethyltestosterone (oxymesterone) from MT cannot be underestimated. Although it is only detected as a minor and short-term excretion metabolite, it cannot be overlooked as it was found in both in vitro and in vivo experiments. The use of a combination of different mass spectrometric instruments allowed reliable conclusions to be reached, and it was shown that special attention must be given to artefact formation.

Methiopropamine is a structural analog of methamphetamine that is categorized as a novel psychoactive substance. It primarily acts as a norepinephrine–dopamine reuptake inhibitor and, secondarily, as a serotonin reuptake inhibitor. In humans, methiopropamine induces stimulation and alertness and increases focus and energy. However, significant side effects are reported, such as tachycardia, anxiety, panic attacks, perspiration, headache, and difficulty in breathing. To date, little data is available regarding its pharmacodynamic effects, thereby we aimed to investigate the acute in vivo effects induced by this drug on sensorimotor responses, body temperature, pain thresholds, motor activity, and cardiovascular and respiratory systems in CD-1 male mice. We selected a range of doses that correspond to the whole range of human reported use, in order to evaluate the threshold of adverse effects presentation. This study demonstrates that methiopropamine acts as a dopaminergic and noradrenergic stimulating drug and that the highest doses (10–30 mg/kg) impair the visual placing response, facilitate the acoustic and tactile response, induce hypothermia, increase mechanical and thermal analgesia, stimulate locomotor activity, induce motor stereotypies, and strongly affected cardiovascular and respiratory parameters, increasing heart rate, breath rate, and blood pressure but reducing oxygen saturation. On the contrary, lower doses do not show any of those effects. We hypothesize that there is a range of doses that do enhance performance but do not seem hazardous to users: this gap could induce the perception of safety and increase the abuser population.

We present the case of a 31-year-old woman who underwent a right surrenectomy for cortical adenoma secreting cortisol responsible of Cushing’s syndrome. After surgery, she was first treated with cortisone acetate 37.5 mg/die and then with hydrocortisone 20 mg/die. Fludrocortisone was administered at the dosage of 0.05 mg/die. Patient complained of severe asthenia and drowsiness and decided to stop therapy. Reduction and subsequent withdrawal from this therapy lasted four months and it took advantage of intramuscular tetracosactide injection. One-year follow-up showed normal values of both basal and stimulated serum cortisol level; the patient was in good physical condition and did not complain any more with asthenia.

Rationale The aromatase inhibitor formestane (4-hydroxyandrost-4-ene-3,17-dione) is included in the World Anti-Doping Agency's List of Prohibited Substances in Sport. However, it also occurs endogenously as do its 2-, 6- and 11-hydroxy isomers. The aim of this study is to distinguish the different isomers using gas chromatography/electron ionization mass spectrometry (GC/EI-MS) for enhanced confidence in detection and selectivity for determination. Methods Established derivatization protocols to introduce [2 H9 ]TMS were followed to generate perdeuterotrimethylsilylated and mixed deuterated derivatives for nine different hydroxy steroids, all with 3-keto-4-ene structure. Formestane was additionally labelled with H2 18 O to obtain derivatives doubly labelled with [2 H9 ]TMS and 18 O. GC/EI-MS spectra of labelled and unlabelled TMS derivatives were compared. Proposals for the generation of fragment ions were substantiated by high-resolution MS (GC/QTOFMS) and tandem mass spectrometry (MS/MS) experiments. Results Subclass-specific fragment ions include m/z 319 for the 6-hydroxy and m/z 219 for the 11-hydroxy compounds. Ions at m/z 415, 356, 341, 313, 269 and 267 were indicative for the 2- and 4-hydroxy compounds. For their discrimination the transition m/z 503 → 269 was selective for formestane. In 2-, 4- and 6-hydroxy steroids loss of a TMSO radical takes place as cleavage of a TMS-derived methyl radical and a neutral loss of (CH3 )2 SiO. Further common fragments were also elucidated. Conclusions With the help of stable isotope labelling, the structures of postulated diagnostic fragment ions for the different steroidal subclasses were elucidated. 18 O-labelling of the other compounds will be addressed in future studies to substantiate the obtained findings. To increase method sensitivity MS3 may be suitable in future bioanalytical applications requiring discrimination of the 2- and 4-hydroxy compounds.

Background: folliculogenesis is a strictly regulated process that may be affected by endocrine disrupting chemicals (EDCs) through sometimes not so clear molecular mechanisms. Methods: we conducted a multicentric observational study involving six fertility centers across Italy, prospectively recruiting 122 women attending a fertility treatment. Recruited women had age ≤42 years, and normal ovarian reserve. Blood and follicular fluid samples were taken for EDCs measurement using liquid chromatography tandem mass spectrometry and each woman completed an epidemiological questionnaire. Results: The main EDCs found were monobutyl phthalate (MBP) (median blood: 8.96 ng/mL, follicular fluid 6.43 ng/mL), monoethylhexyl phthalate (MEHP) (median blood: 9.16 ng/mL, follicular fluid 7.68 ng/mL) and bisphenol A (BPA) (median blood: 1.89 ng/mL, follicular fluid 1.86 ng/mL). We found that serum MBP concentration was significantly associated with the considered area (p < 0.001, adj. mean: 7.61 ng/mL, 14.40 ng/mL, 13.56 ng/mL; Area 1: Milan–Turin, Area 2: Rome–Naples; Area 3: Catania–Bari, respectively) but negatively with home plastic food packaging (p = 0.004). Follicular MBP was associated with irregular cycles (p = 0.019). No association was detected between EDCs and eating habits and other clinical and epidemiological features. Conclusions: This study represents the first Italian biomonitoring of plastic EDCs in follicular fluid, laying the basis for future prospective evaluation on oocyte quality before assisted reproduction techniques (ART).

Γ-Valero-lactone (GVL) is a freely marketed organic solvent and food additive. GVL is also an underrated legal substitute for γ-hydroxybutyric acid (GHB), and it is sold under the trade name "Tranquili-G". GVL is usually not included in forensic drug testing, even though it has been reported in drug-facilitated sexual assaults (DFSA). To date, few studies verified the effectiveness of GVL as a hypnotic/narcotic substance, and no data are available on its urinary excretion profile. This work aims to fill this knowledge gap and lead to the introduction of GVL in forensic drug testing, especially when DFSA is suspected. To monitor the timeframe of GVL activity in vivo, we have assessed the effects on sensorimotor responses in visual placing tests carried out on CD-1 mice at a dose of 400 mg/kg of GVL, administered by gastric gavage. In parallel, samples of the in vitro and in vivo metabolism studies were analyzed using a rapid and cost-effective analytical procedure based on gas chromatography coupled to mass spectrometry. Our data confirmed that GVL impairs visual placing response in mice in the first 4 hours after the intake, and they also show that GVL is a less potent substitute of GHB. The urinary excretion profile of GVL is consistent with the results of the behavioral study, with a maximum of excretion in the first 5 hours after the intake. GVL is only detectable in the first 0-8 hours after the intake. Our data confirmed the same rapid urinary excretion rate of GHB in urine and the related forensic implications, with the risk of possible false-negative results, especially when DFSA is suspected.

The potential consequences of drug-drug interaction on the strategies adopted by anti-doping laboratories to report an adverse analytical finding for morphine were investigated. We evaluated in vitro the effects of 14 drugs on the principal metabolic pathways of morphine. The selected drugs are among those most commonly used by the athletes, none of them presently included in the World Anti-Doping Agency (WADA) Prohibited List. The non-prohibited drugs included 4 antifungals (fluconazole, itraconazole, ketoconazole, and miconazole), 6 benzodiazepines (alprazolam, bromazepam, clonazepam, lorazepam, lormetazepam, and triazolam), and 4 non-steroidal anti-inflammatory drugs (diclofenac, ibuprofen, ketoprofen, and nimesulide). The in vitro assays were based on the use of either human liver microsomes or uridine 5'-diphospho-glucuronosyl-transferases. Morphine and its glucuronides were determined by developed liquid chromatography-mass spectrometry procedure after dilution with an aqueous solution containing their deuterated isotopologues as internal standards. Morphine is mainly excreted as phase II metabolites: about 70% of the parent compound is found to be biotransformed by UGT2B7 to morphine-3-glucuronide (6065%) and morphine-6-glucuronide (5-10%). A reduction of the enzymatic activity of the UGT2B7 was recorded in the presence of 9 of the 14 drugs under investigation (ketoconazole, miconazole, itraconazole, diclofenac, ibuprofen, clonazepam, lorazepam, lormetazepam, and triazolam), with a consequent significant reduction of the levels of the glucuronide metabolites. This phenomenon in vivo may affect the rate of the urinary excretion of morphine with the risk of reporting "false negative" results, especially in case of results close to the decision limit value set by WADA.

Background The outstanding performance of an elite athlete might be associated with changes in their blood metabolic profile. The aims of this study were to compare the blood metabolic profiles between moderate- and high-power and endurance elite athletes and to identify the potential metabolic pathways underlying these differences. Methods Metabolic profiling of serum samples from 191 elite athletes from different sports disciplines (121 high- and 70 moderate-endurance athletes, including 44 high- and 144 moderate-power athletes), who participated in national or international sports events and tested negative for doping abuse at anti-doping laboratories, was performed using non-targeted metabolomics-based mass spectroscopy combined with ultrahigh-performance liquid chromatography. Multivariate analysis was conducted using orthogonal partial least squares discriminant analysis. Differences in metabolic levels between high- and moderate-power and endurance sports were assessed by univariate linear models. Results Out of 743 analyzed metabolites, gamma-glutamyl amino acids were significantly reduced in both high-power and high-endurance athletes compared to moderate counterparts, indicating active glutathione cycle. High-endurance athletes exhibited significant increases in the levels of several sex hormone steroids involved in testosterone and progesterone synthesis, but decreases in diacylglycerols and ecosanoids. High-power athletes had increased levels of phospholipids and xanthine metabolites compared to moderate-power counterparts. Conclusions This pilot data provides evidence that high-power and high-endurance athletes exhibit a distinct metabolic profile that reflects steroid biosynthesis, fatty acid metabolism, oxidative stress, and energy-related metabolites. Replication studies are warranted to confirm differences in the metabolic profiles associated with athletes’ elite performance in independent data sets, aiming ultimately for deeper understanding of the underlying biochemical processes that could be utilized as biomarkers with potential therapeutic implications. Electronic supplementary material The online version of this article (10.1186/s40798-017-0114-z) contains supplementary material, which is available to authorized users.

4,4’-Dimethylaminorex (4,4’-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±)cis (0.1–60 mg/kg) and (±)trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±)cis at 1, 10 or 60 mg/kg + (±)trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4′-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±)cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±)trans stereoisomer was ineffective whilst the stereoisomers’ co-administration resulted in a worsening of the toxic (±)cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±)trans stereoisomer reduced the metabolism of the (±)cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.

The urinary excretion profile of prednisolone and prednisone after both systemic (i.e., oral) and topical (i.e., ocular and intranasal) administration was studied by liquid chromatography coupled to mass spectrometry, also to select the most appropriate marker(s) of intake for doping control purposes. Urines were collected from ten subjects every 3 h before and after the administration of therapeutic doses of pharmaceutical formulations containing either prednisone or prednisolone. Samples were subjected to enzymatic hydrolysis (performed for the investigation on the glucuronide profile) followed by liquid/liquid extraction with tert-butylmethylether in alkaline conditions. The chromatographic separation was carried out on C18 column, employing as mobile phases ultrapurified water and acetonitrile, both containing 0.1% of formic acid. Detection was achieved using as mass spectrometric analyzer a triple quadrupole, with positive ion electrospray ionization and multiple reaction monitoring as acquisition mode. After both systemic and topical use, the compounds excreted in urine in higher concentration were prednisone, prednisolone and 20β-dihydro-prednisolone followed by 20α-dihydro-prednisolone and 20α/β-dihydro-prednisone. All were excreted mainly as unconjugated compounds, with a maximum of excretion in the first 3-9 h after the administration. After systemic use, prednisone and prednisolone were both detectable for at least 24 h in concentrations ranging from 5 to 500 ng/mL and from 5 to 900 ng/mL respectively. Whereas, after topical administration, prednisone and prednisolone were detectable for at least 18 h in concentrations ranging from 5 to 140 ng/mL and from 5 to 50 ng/mL respectively.

Prednisone and prednisolone are two anti-inflammatory steroidal drugs listed by the World Anti-Doping Agency (WADA) within the class of glucocorticoids, which are prohibited "in competition" and when administered systemically. Their presence in collected urine samples may be attributed, if no exogenous administration has occurred, to an in situ microbial formation from endogenous steroids. In this work, a gas chromatography coupled to carbon isotope ratio mass spectrometry (GC-C-IRMS) method was developed and validated to distinguish an exogenous origin from an endogenous one. Eight prednisone/prednisolone pharmaceutical preparations commercially available in Italy were analysed to establish an exogenous δ13 C value reference range (-28.96 ± 0.39‰). No more than 25 mL of urine was processed and no derivatization nor intentional steroids structure modifications were performed before the GC-C-IRMS analysis. A first HPLC purification step was set up to isolate the three endogenous reference compounds (ERCs) selected (tetrahydro-11-deoxycortisol (THS), pregnanediol (PD), and pregnanetriol (PT)), while a second LC purification was necessary to separate prednisone from prednisolone. In the GC-C-IRMS analysis, two different GC run methods were set up to guarantee better sensitivity and selectivity for each compound. Both prednisone and prednisolone showed signals (m/z 44) with amplitudes within the method linearity range to a lower urinary concentration of 20 ng/mL (< WADA reporting level, 30 ng/mL). The method was fully validated according to WADA requirements. As a proof of concept, urine samples collected from two excretion studies in healthy male volunteers, after a prednisone or prednisolone administration, were analysed by the proposed method, demonstrating its applicability for the analysis of real samples.

Cytochrome P450 enzymes (CYPs) are capable of catalyzing regio- and stereo-specific oxy functionalization reactions, which otherwise are major challenges in organic chemistry. In order to make the best possible use of these biocatalysts it is imperative to understand their specificities. Human CYP21A2 (steroid 21-hydroxylase) acts on the side-chain attached to C-17 in ring D of a steroid substrate, but the configuration of ring A also plays a prominent role in substrate cognition. Here, we comprehensively investigated this relationship using sixteen 17,17-dimethyl-18-nor-13-ene steroids with different arrangements of hydroxy-, oxo-, fluoro- and chloro-groups and in the presence or absence of double bonds (Δ1 and/or Δ4) and heteroatoms in ring A. The results show that presence of a 3-oxo group is a strict requirement for a CYP21A2 substrate, while the other configurations tested were all tolerated. This was also confirmed by control experiments using endogenous steroids. While progesterone and 17-hydroxyprogesterone were hydroxylated at C-21, (17-hydroxy-) pregnenolone did not react. Molecular docking experiments indicate that the interaction of the carbonyl group at C-3 to the side-chain Arg234 of the enzyme is indispensable.

Recent studies suggest that the anabolic effect of ecdysterone, a naturally occurring steroid hormone claimed to enhance physical performance, is mediated by estrogen receptor (ER) binding. In comparison with the prohibited anabolic agents (e.g., metandienone and others), ecdysterone revealed to be even more effective in a recent study performed in rats. However, scientific studies in humans are very rarely accessible. Thus, our project aimed at investigating the effects of ecdysterone-containing products on human sport exercise. A 10-week intervention study of strength training of young men (n = 46) was carried out. Different doses of ecdysterone-containing supplements have been administered during the study to evaluate the performance-enhancing effect. Analysis of blood and urine samples for ecdysterone and potential biomarkers of performance enhancement has been conducted. To ensure the specificity of the effects measured, a comprehensive screening for prohibited performance-enhancing substances was also carried out. Furthermore, the administered supplement has been tested for the absence of anabolic steroid contaminations prior to administration. Significantly higher increases in muscle mass were observed in those participants that were dosed with ecdysterone. The same hypertrophic effects were also detected in vitro in C2C12 myotubes. Even more relevant with respect to sports performance, significantly more pronounced increases in one-repetition bench press performance were observed. No increase in biomarkers for liver or kidney toxicity was noticed. These data underline the effectivity of an ecdysterone supplementation with respect to sports performance. Our results strongly suggest the inclusion of ecdysterone in the list of prohibited substances and methods in sports in class S1.2 “other anabolic agents”.

Intensive exercise of elite athletes can lead to physiological alterations in the cardiovascular system in response to increased stroke volume and blood pressure, known collectively as cardiovascular demand (CD). This study aimed to compare metabolic differences in elite athletes with high vs low/moderate CD and to reveal their underlying metabolic pathways as potential biomarker signatures for assessing health, performance, and recovery of elite athletes. Metabolic profiling of serum samples from 495 elite athletes from different sport disciplines (118 high CD and 377 low/moderate CD athletes) was conducted using non-targeted metabolomics-based mass spectroscopy combined with ultra-high-performance liquid chromatography. Results show that DAGs containing arachidonic were enriched in high CD together with branched-chain amino acids, plasminogens, phosphatidylcholines, and phosphatidylethanolamines, potentially indicating increased risk of cardiovascular disease in the high CD group. Gamma-glutamyl amino acids and glutathione metabolism were increased in low/moderate CD group, suggesting more efficient oxidative stress scavenging mechanisms than the high CD group. This first most comprehensive metabolic profiling of elite athletes provides an evidence that athletes with different CD show a unique metabolic signature that reflects energy generation and oxidative stress and potentially places the high CD group at a higher risk of cardiovascular disease. Further studies are warranted for confirmation and validation of findings in other sport groups in light of potential confounders related to limited available information about participants. This study was funded by Qatar National Research Fund (QNRF), Grant number NPRP7‐272‐1‐041 (MAE, KS, CG and FB).

Urine collection containers used in the doping control collection procedure do not provide a protective environment for urine, against degradation by microorganisms and proteolytic enzymes. An in-house chemical stabilization mixture was developed to tackle urine degradation problems encountered in human sport samples, in cases of microbial contamination or proteolytic activity. The mixture consists of antimicrobial substances and protease inhibitors for the simultaneous inactivation of a wide range of proteolytic enzymes. It has already been tested in lab-scale, as part of World Anti-Doping Agency's (WADA) funded research project, in terms of efficiency against microbial and proteolytic activity. The present work, funded also by WADA, is a follow-up study on the improvement of chemical stabilization mixture composition, application mode and limitation of interferences, using pilot urine collection containers, spray-coated in their internal surface with the chemical stabilization mixture. Urine in plastic stabilized collection containers have been gone through various incubation cycles to test for stabilization efficiency and analytical matrix interferences by three WADA accredited Laboratories (Athens, Ghent, and Rome). The spray-coated chemical stabilization mixture was tested against microorganism elimination and steroid glucuronide degradation, as well as enzymatic breakdown of proteins, such as intact hCG, recombinant erythropoietin and small peptides (GHRPs, ipamorelin), induced by proteolytic enzymes. Potential analytical interferences, observed in the presence of spray-coated chemical stabilization mixture, were recorded using routine screening procedures. The results of the current study support the application of the spray-coated plastic urine container, in the doping control collection procedure. Copyright © 2016 John Wiley & Sons, Ltd.

Liver in vitro systems that allow reliable prediction of major human in vivo metabolic pathways have a significant impact in drug screening and drug metabolism research. In the present study, a novel porous scaffold composed of alginate was prepared by employing a gas-in-liquid foaming approach. Galactose residues were introduced on scaffold surfaces to promote cell adhesion and to enhance liver specific functions of the entrapped HepG2/C3A cells. Hepatoma cells in the gal-alginate scaffold showed higher levels of liver specific products (albumin and urea) and were more responsive to specific inducers (e.g. dexamethasone) and inhibitors (e.g. ketoconazole) of the CYP3A4 system than in conventional monolayer culture. HepG2/C3A cells were also more efficient in terms of rapid elimination of testosterone, used as a model substance, at rates comparable to those of in vivo excretion. In addition, an improvement in metabolism of testosterone, in terms of phase II metabolite formation, was also observed when the more differentiated HepaRG cells were used. Together the data suggest that hepatocyte/gas templated alginate-systems provide an innovative high throughput platform for in vitro drug metabolism and drug-drug interaction studies, with broad fields of application, and might provide a valid tool for minimizing animal use in preclinical testing of human relevance.

Glucocorticoids are included in the S9 section of the World Anti-doping Agency (WADA) prohibited list international standard. Some among them are pseudo-endogenous steroids, like cortisol and cortisone, which present the same chemical structure as endogenously produced steroids. We are proposing an analytical method based on gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) which allows discrimination between endogenous and synthetic origin of the urinary metabolites of the pseudo-endogenous glucocorticoids. A preliminary purification treatment by high-performance liquid chromatography (HPLC) of the target compounds (TC) (i.e., cortisol, tetrahydrocortisone (THE) 5α-tetrahydrocortisone (aTHE), tetrahydrocortisol (THF), and 5α-tetrahydrocortisol (aTHF)) allows collection of extracts with adequate purity for the subsequent analysis by IRMS. A population of 40 urine samples was analyzed for the TC and for the endogenous reference compounds (ERC: i.e., 11-desoxy-tetrahydrocortisol (THS) or pregnanediol). For each sample, the difference between the delta values of the ERCs and TCs (Δδ values) were calculated and based on that, some decision limits for atypical findings are proposed. The limits are below 3% units except for cortisol. The fit to purpose of the method has been confirmed by the analysis of urine samples collected in two patients under treatment with 25 mg of cortisone acetate (p.o). The samples showed Δδ values higher than 3 for at least 24 h following administration depending on the TC considered. The method can easily be integrated into existing procedures already used for the HPLC purification and IRMS analysis of pseudo-endogenous steroids with androgenic/anabolic activity.

In this work, we present a gas-chromatography tandem mass spectrometry (GC-MS/MS) method for the identification of the sulfo-conjugate metabolites of pseudo-endogenous steroids (endogenous steroids when administered exogenously). We have preliminarily evaluated the performances of different preparations of sulfatases from Pseudomonas aeruginosa and Helix pomatia, characterized by various origins and catalytic activities, and compared the efficacy of the enzymatic hydrolysis with chemical hydrolysis, performed with a mixture of ethyl acetate, methanol, and sulphuric acid. A procedure for the selective isolation of steroid conjugates from the urine matrix has been designed and optimized, based on the "sequential" extraction of the glucuro-conjugated and of the sulfo-conjugated fractions, performed by two different direct methods, i.e. by ion paired extraction or solid-phase extraction. More specifically, the former method is based on the use of N,N-dimethylephedrinium bromide as the ion paired extraction reagent, while the latter on the use of WAX® (weak anion exchange) cartridges. The performance of the newly developed procedure has been assessed by the analysis of real urine excretion samples collected after the oral intake of a single dose of dehydroepiandrosterone (DHEA) or androstenedione (AED), measuring the concentration of epiandrosterone (EpiA) sulfate. Our results have shown the following: (i) although the yields of chemical hydrolysis and enzymatic hydrolysis are in some cases quite similar, the former is generally preferable since it results in the quantitative cleavage of sulfate moiety; (ii) ion paired extraction has been selected as the most reliable method for direct isolation of sulfate steroids from urine matrices; (iii) EpiA sulfate allows to prolong the detectability of DHEA and AED when compared to routinely used steroidal target compounds.

Formestane (4-hydroxyandrost-4-ene-3,17-dione, 4OH-AED) is an aromatase inhibitor prohibited in sports. In recent years, it has been demonstrated that it can also originate endogenously by the hydroxylation in C4 position of androstenedione. Thus, the use of isotope ratio mass spectrometry (IRMS) is mandatory according to the World Antidoping Agency (WADA) to discriminate endogenous from synthetic origin. In a previous work and after oral administrations of formestane (4OH-AED), the ratio between the main formestane metabolite (4α-hydroxyepiandrosterone; 4OH-EA) and formestane parent compound could help to identify the endogenous origin, avoiding unnecessary and costly IRMS confirmations. In the present work, we investigated whether the same criteria could also be applied after transdermal applications. Six volunteers were transdermally treated once with formestane. Urine samples were collected for 120 h postadministration and analyzed by gas chromatography coupled to mass spectrometry (GC–MS and GC–MS/MS). Formestane and its major metabolites were monitored. The kinetic profile of formestane and its main metabolites was found different between oral and transdermal application. A shift on the excretion of the metabolites compared to formestane itself that can be observed after the oral administration, is absent after the transdermal one. This makes that a simple criteria cannot be applied to differentiate the endogenous from the synthetic origin based on metabolic ratios. The ratio between 4-hydroxyepiandrosterone and 4-hydroxyandrosterone (4OH-A) can be used to differentiate the route of administration. Ratios higher than one (4OH-EA/4OH-A > 1) are diagnostic of an oral administration. This allows to correctly interpret the 4OH-EA/4OH-AED ratio as proposed in our previous investigation. The results of this work demonstrate that the use of appropriate biomarkers (metabolic ratios) helps to reach correct conclusions without using complex and costly instrumentation approaches.

Testosterone is one of the most abused pseudo-endogenous anabolic steroids in sport doping. The current method adopted to detect the abuse of testosterone and other pseudo-endogenous steroids (endogenous steroids when administered exogenously) is first based on the longitudinal monitoring of several urinary biomarkers, which constitute the so called “steroidal module” of the Athlete Biological Passport (ABP): atypical samples undergo a confirmation analysis based on the measurement of the 13C/12C isotopic ratio of selected target compounds, to distinguish their endogenous or exogenous origin. At the same time, testosterone administration can be allowed in athletes diagnosed with hypogonadism, provided they are granted a therapeutic use exemption by the relevant medical authority. In this pilot study we have investigated whether the approach based on the preliminary determination of the urinary steroid profile, in the format considered in the steroidal module of the ABP, also integrated with the inclusion of the sulfo-conjugates and of additional target steroids, can retain its validity also in the case of hypogonadal athletes. We have studied the effects of a single low dose (40 mg) of testosterone gel (T-gel) on the urinary concentration of the markers of steroidal module of the ABP, as well as on some additional steroid markers. The study was based on the analysis of urinary samples from 19 non-hospitalized hypogonadal men, 10 of them with late-onset hypogonadism (LOH), collected before, after 4 h and after 24 h the transdermal self-administration of 40 mg of T-gel. None of the patient had any co-morbidities possibly affecting the urinary excretion of the steroidal markers. The steroidal markers were quantified by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) after the enzymatic hydrolysis of the respective glucuro-conjugates and the chemical hydrolysis of the respective sulfo-conjugates. Targeted GC-MS/MS analysis was carried out operating in electron impact (EI) ionization mode, with acquisition in multiple reaction monitoring (MRM) mode. Our preliminary results show that, as expected, the treatment with T-gel leads, in all hypogonadal men, to an increase of the urinary concentration of the glucuro-conjugate metabolites of testosterone and its main metabolites, with special relevance to those with 5α-reduction. Furthermore, samples collected from non-LOH hypogonadal men showed an increase also in the levels of epitestosterone glucuronide, testosterone sulfate and epitestosterone sulfate. Apart from their biochemical and pharmacological relevance, these outcomes could be leveraged to refine the analytical strategy currently followed in the antidoping field for the analysis of the urinary steroidal markers, with potential implications also in other forensic and/or clinical investigations.

The reactions involved in the metabolic pathways of SR9009 were characterized by liquid chromatography-mass spectrometry (LC-MS) to identify the most appropriate marker(s) of use. The effects of gender, genetic polymorphism, and drug-drug interaction on the metabolic profile of SR9009 were also evaluated. In vitro approaches were based on the use of human liver microsomes and cytochrome P450 isoforms. Sample preparation included an enzymatic hydrolysis (performed only for the phase II investigation) followed by liquid-liquid extraction. The chromatographic separation was carried out using a reverse-phase column; detection was performed by either a triple-quadrupole or a time-of-flight system in positive electrospray ionization and different acquisition modes. In the presence of human liver microsomes, SR9009 was biotransformed to 13 metabolites by CYP3A4, CYP3A5, CYP2C19, and CYP2D6 isoenzymes. The reactions included hydroxylation, de-alkylation, oxidation, and combinations thereof, the de-alkylated metabolites being the most abundant. Once formed the mentioned metabolites underwent glucuronidation. Concerning the effects of gender, genetic polymorphism, and drug-drug interaction on the metabolic profile of SR9009, our observation have shown the following: (a) No significant alterations were measured between female and male, (b) significant differences were registered using either the CYP2D6 or CYP2C19 allelic variants, and finally (c) significant alterations were registered in the presence of ketoconazole, miconazole, fluoxetine, nefazodone and paroxetine; moderate variation were instead registered with fluconazole, itraconazole, gestodene, and levonorgestrel. This observation put in evidence the importance to take into account both genetic polymorphism and drug-drug interaction to select the most appropriate marker(s) of use in doping analysis.

Anectodical information suggests that flavonoids may be widely used among athletes for their multiple biochemical and pharmacological effects. We have evaluated in vitro the effects of two synthetic isoflavones, methoxyisoflavone and ipriflavone, on the catalytic activity of human aromatase (CYP19), the enzyme catalyzing the conversion of androgens (ie, testosterone or androstenedione) to estrogens (ie, estradiol and estrone). The potential inhibitory effect was evaluated by measuring the rate of aromatization of testosterone, monitored by gas chromatography-mass spectrometry (GC-MS), both in the presence and in the absence of methoxyisoflavone or ipriflavone, comparing their effects with those of synthetic aromatase inhibitors (formestane, anastrozole, and aminoglutethimide) presently included in the list of prohibited substances and methods, and of natural flavonoids (chrysin, quercetin, and daidzein), that are known inhibitors of CYP19. The preliminary results of our in vitro study show that methoxyisoflavone and ipriflavone act as competitive inhibitors of aromatase, the degree of inhibition measured in vitro being of the same order of magnitude of that of the aromatase inhibitors commonly used in anti-estrogenic therapies. Our preliminary in vitro results indicate that, in principle, a sufficiently large intake of isoflavones could alter the kinetics of the dynamic equilibria between androgens and estrogens, suggesting their monitoring in doping control routine analysis.

Background Supplements are widely used among elite athletes to maintain health and improve performance. Despite multiple studies investigating use of dietary supplements by athletes, a comprehensive profiling of serum supplement metabolites in elite athletes is still lacking. This study aims to analyze the presence of various xenobiotics in serum samples from elite athletes of different sports, focusing on metabolites that potentially originate from nutritional supplements. Methods Profiling of xenobiotics in serum samples from 478 elite athletes from different sports (football, athletics, cycling, rugby, swimming, boxing and rowing) was performed using non-targeted metabolomics-based mass spectroscopy combined with ultrahigh-performance liquid chromatography. Multivariate analysis was performed using orthogonal partial least squares discriminant analysis. Differences in metabolic levels among different sport groups were identified by univariate linear models. Results Out of the 102 detected xenobiotics, 21 were significantly different among sport groups including metabolites that potentially prolong exercise tolerance (caffeic acid), carry a nootropic effect (2-pyrrolidinone), exert a potent anti-oxidant effect (eugenol, ferulic acid 4 sulfate, thioproline, retinol), or originate from drugs for different types of injuries (ectoine, quinate). Using Gaussian graphical modelling, a metabolic network that links various sport group-associated xenobiotics was constructed to further understand their metabolic pathways. Conclusions This pilot data provides evidence that athletes from different sports exhibit a distinct xenobiotic profile that may reflect their drug/supplement use, diet and exposure to various chemicals. Because of limitation in the study design, replication studies are warranted to confirm results in independent data sets, aiming ultimately for better assessment of dietary supplement use by athletes.